TREK-1 Localization in Trabecular Meshwork by Immunohistochemistry Protocol
Phase 1: Tissue Collection and Processing (Days 1-7)
Human Trabecular Meshwork Acquisition: Obtain human donor eyes (n=12, ages 50-75, both sexes) from eye banks within 24 hours of death. Enucleate eyes, rinse in sterile PBS, and dissect trabecular meshwork (TM) tissue from the iridocorneal angle. Flash-freeze 4 tissue blocks in OCT compound for cryosectioning (8 μm thickness). Fix remaining tissue in 4% PFA (4°C, 4 hours) for paraffin embedding (5 μm sections).
Quality Control: Verify tissue integrity via H&E staining. Exclude samples with >20% epithelial disruption or significant autolysis. Store slides at -80°C until use.
...TREK-1 Localization in Trabecular Meshwork by Immunohistochemistry Protocol
Phase 1: Tissue Collection and Processing (Days 1-7)
Human Trabecular Meshwork Acquisition: Obtain human donor eyes (n=12, ages 50-75, both sexes) from eye banks within 24 hours of death. Enucleate eyes, rinse in sterile PBS, and dissect trabecular meshwork (TM) tissue from the iridocorneal angle. Flash-freeze 4 tissue blocks in OCT compound for cryosectioning (8 μm thickness). Fix remaining tissue in 4% PFA (4°C, 4 hours) for paraffin embedding (5 μm sections).
Quality Control: Verify tissue integrity via H&E staining. Exclude samples with >20% epithelial disruption or significant autolysis. Store slides at -80°C until use.
Phase 2: Immunohistochemical Staining (Days 8-14)
Antigen Retrieval: Deparaffinize slides in xylene (2× 5 min), rehydrate through graded ethanol series. Perform heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0, 95°C, 20 min). Cool slides to room temperature, wash 3× in PBS.
Primary Antibody Incubation: Block in 5% normal donkey serum (1 hour, RT). Incubate with primary antibodies: rabbit anti-TREK-1 (1:100, Alomone Labs #ANT-012) and mouse anti-GBF2 (Golgi marker, 1:200, 1 hour, RT). Include isotype controls (rabbit IgG, mouse IgG1). Wash 3× PBS.
Secondary Antibody and Detection: Apply donkey anti-rabbit Alexa Fluor 594 (1:500, 1 hour, RT) and donkey anti-mouse Alexa Fluor 488 (1:500, 1 hour, RT). Counterstain nuclei with DAPI (300 nM, 5 min). Mount in ProLong Gold antifade reagent. Image within 48 hours.
Phase 3: Confocal Imaging and Quantitative Analysis (Days 15-21)
Microscopy: Image on Leica SP8 confocal microscope (63× oil immersion objective, NA 1.4). Acquire z-stacks (0.5 μm steps, 20 slices per field) from 3 non-overlapping TM regions per slide. Set laser power and gain to avoid saturation in isotype controls.
Quantification Protocol: Using ImageJ/Fiji: (1) segment TREK-1+ cells based on signal threshold (3× background), (2) define subcellular compartments (membrane, cytoplasm, nucleus) using marker-based masks, (3) measure mean fluorescence intensity (MFI) per compartment. Calculate membrane-to-cytoplasm ratio for each cell (n≥50 cells per sample).
Statistical Analysis: Compare TREK-1 membrane localization between glaucoma and normal donor eyes. Use Mann-Whitney U test for non-parametric comparison (primary outcome: membrane MFI). Correlate with donor age and post-mortem time as covariates.